Sporophyte Directed Gametogenesis via the Ubiquitin Proteasome System

Sporophyte Directed Gametogenesis via the Ubiquitin Proteasome System | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sporophyte Directed Gametogenesis via the Ubiquitin Proteasome System View ORCID Profile Prakash Sivakumar , Saurabh Pandey , A Ramesha , Jayeshkumar Narsibhai Davda , Aparna Singh , Chandan Kumar , Hardik Gala , Veeraputhiran Subbiah , Harikrishna Adicherla , Jyotsna Dhawan , L. Aravind , Imran Siddiqi doi: https://doi.org/10.1101/2024.12.23.630054 Prakash Sivakumar 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad - 201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Prakash Sivakumar Saurabh Pandey 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site A Ramesha 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jayeshkumar Narsibhai Davda 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aparna Singh 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chandan Kumar 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hardik Gala 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Veeraputhiran Subbiah 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Harikrishna Adicherla 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jyotsna Dhawan 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site L. Aravind 3 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health ; Bethesda, MD 20894, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Imran Siddiqi 1 CSIR–Centre for Cellular and Molecular Biology ; Uppal Road, Hyderabad 500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad - 201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: imran{at}ccmb.res.in Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Plants alternate between diploid sporophyte and haploid gametophyte generations. In mosses which retain features of ancestral land plants, the gametophyte is dominant and has an independent existence. However, in flowering plants the gametophyte has undergone evolutionary reduction to just a few cells enclosed within the sporophyte. The gametophyte is thought to retain genetic control of its development even after reduction. Here we demonstrate that male gametophyte development in Arabidopsis, long considered to be autonomous, is also under genetic control of the sporophyte via a repressive mechanism involving large-scale regulation of protein turnover. We identify an Arabidopsis gene SHUKR as an inhibitor of male gametogenesis. SHUKR is unrelated to proteins of known function and acts sporophytically in meiosis to control gametophyte development by negatively regulating expression of a large set of ubiquitination genes specific to post-meiotic gametogenesis. This control is late-emerging as SHUKR homologs are found only in eudicots. We show that SHUKR is rapidly evolving under positive selection suggesting that variation in control of protein turnover during male gametogenesis has played an important role in evolution within eudicots. Introduction Alternation between two distinct generations: a diploid sporophyte and a haploid gametophyte is a central feature of the land plant life cycle 1 – 3 . Land plants likely evolved from an algal ancestor related to extant charophycean algae which comprise the closest relatives to land plants 4 , 5 . Charophycean algae have a predominantly haploid life cycle, the diploid phase being transient as the zygote directly undergoes meiosis to give haploid spores. The emergence of a multicellular diploid sporophyte contributed to reproductive success on land by increasing the number of spores produced from a single fertilization event, and also increased potential for genetic variability and evolutionary change 1 . From a simple ancestral body plan, land plants comprising bryophytes (mosses, liverworts, and hornworts) and vascular plants evolved progressively specialized cell types, organs, and architectures adapted for life on land. These acquisitions have been based on development and increase in complexity of a multicellular sporophyte 6 , 7 . In vascular plants the sporophyte evolved to become the dominant phase and the gametophyte has reduced. In flowering plants the gametophyte is reduced to just a few cells and develops enclosed within the sporophyte 8 ( Fig. 1a ). The differing trajectories taken by the sporophyte and gametophyte in land plant evolution reflects an uncoupling between their body plan evolution consistent with different genetic programs directing their body organization 7 , 9 , 10 , 11 , 12 . Download figure Open in new tab Fig. 1: SHUKR is required for pollen development. (a) Schematic showing reduction of the gametophyte in flowering plants. (b) Sterility (short siliques) and reduced pollen viability (green staining) in skr , and complementation by a genomic SKR transgene. (c) Tables showing reduced seed set, male-specific sterility, and recessive Mendelian segregation for skr ; numbers in brackets are those expected for a 3:1 ratio and the χ 2 test p value. (d) RT-PCR analysis of SKR expression in vegetative and reproductive tissues. (e) GUS staining for proSKR:GFP-GUS transgene expression in successive unopened buds. (f) GUS staining for proSKR:GFP-GUS expression in meiotic stage anthers. (g) anti-GFP antibody staining for proSKR:GFP-GUS expression in male meiosis and microspores. (h) Normal tetrad formation in skr . Scale bar: (e) 0.5 mm, (f) 20 µm. Formation of a germline is an early step in animal development 13 , 14 , however in plants the formation of germ cell precursors takes place late in development 15 . The fact that bryophytes have a multicellular independent gametophyte which produces gametes following extensive mitotic divisions allows overall assignment of the germline to the gametophyte generation 16 . In flowering plants the assignment of the germline is more difficult because of extreme reduction of the gametophyte and its development enclosed within the sporophyte 15 , 17 , 18 . Evidence from comparative evolution showing functional conservation of transcription factors controlling early steps in germ cell development between the liverwort Marchantia polymorpha and Arabidopsis argues for the male germline residing in the gametophyte 16 . Asymmetric cell division of the haploid microspore in Arabidopsis at pollen mitosis I forms a generative cell (GC) and vegetative cell (VC) 16 , 18 . In Arabidopsis, specification of the GC requires bHLH transcription factors, BNB1/BNB2 and DROP1/DROP2 which act in the gametophyte and are orthologous to MpBNB and MpDROP respectively, that also act in the gametophyte to direct gametangia specification and differentiation in Marchantia 19 , 20 . Sperm differentiation is controlled by gametophytic action of the bHLH transcription factor DUO1 in both Arabidopsis and Marchantia 21 , 22 . On the female side gametophytically acting transcription, splicing, and signaling related factors are important for cell specification and cellularization in Arabidopsis female gametophytes 23 – 29 . Therefore, the gametophyte in flowering plants has been considered to largely retain genetic control of its development even though it lacks an independent existence. The extreme reduction of the gametophyte and its development enclosed within the sporophyte provided a “functionally unitary individual” for natural selection 30 , addressing the sensitive requirements for fertilization and reproductive development in a land environment. Formation and function of meiotic cells depends on a range of cellular interactions in the sporophyte 8 , 31 – 33 . These interactions involve transcriptional regulators acting nonautonomously 34 – 36 , cell surface receptor signaling 37 – 39 , and small RNA pathways 36 , 40 , 41 together with phytohormone effectors 42 . Small RNAs 43 and the phytohormone auxin 44 , 45 have also been shown to mediate developmental interactions between cells within gametophytes. The tapetum is a sporophytic nurse tissue surrounding male meiocytes and provides enzymes for release of microspores and components for synthesis of the protective secondary outer coat of the pollen grain, the exine 46 , 47 . Mutants deficient in tapetal function manifest defects in exine formation and lead to pollen abortion 48 – 50 . The tapetum also produces small RNAs that direct DNA methylation of transposons and selected genes in male meiosis contributing to genome integrity and paternal epigenetic inheritance 51 . Studies have shown that isolated uninucleate microspores can form functional pollen in vitro suggesting their development into pollen is autonomous 52 , 53 . It remains unclear whether genetic controls during meiosis broadly regulate postmeiotic gametophyte development. A challenge in uncovering such regulation has been the existence of very many sporophytic mutants that affect core meiosis functions including meiotic recombination and chromosome organization 54 , 55 or else development of somatic cells within the anther or ovule 56 – 59 . These mutants have direct or indirect effects on gametophyte development and show spore or gametophyte abortion, making it difficult to distinguish from mutants affecting meiotic regulation of postmeiotic gametogenesis. Here we report the discovery in Arabidopsis that following evolutionary reduction in cell number of the gametophyte, the sporophyte within the eudicot lineage has gone on to evolve a process established in meiosis under control of the SHUKR gene, by which it directs male gametophyte development. The shukr mutant shows normal meiosis but postmeiotic development is defective. This process controlled by SHUKR involves large-scale control of regulated protein turnover in the developing gametophyte and is under rapid evolution with positive selection. Our findings suggest that the sporophyte in addition to providing supportive conditions for gametogenesis and fertilization, also exerts genetic control in meiosis of postmeiotic male gametogenesis. This control is part of a process that is capable of generating variation in the gametophyte during evolution, which has adaptive value. Results SHUKR is required for male fertility We identified the SHUKR gene in a screen for novel meiotic functions based on coexpression with known meiotic genes in Arabidopsis (see Methods). Three independent insertion lines having insertions in At2g38690 showed male sterility ( Fig. 1b , Extended Data Fig. 1a ). Allelism tests indicated the mutants to be allelic. Anthers from plants homozygous for the insertion contained a highly reduced number of viable pollen, resulting in sterility ( Fig. 1b, c ). Use of wild type pollen as the male donor in crosses to mutant plants resulted in full seed set indicating that female fertility was unaffected ( Fig. 1c ). The male sterile phenotype was inherited as a single gene recessive trait ( Fig. 1c , Extended Data Fig. 1c ). We named the At2g38690 gene SHUKR ( SKR ) after the Sanskrit word Shukranu (/śukrāṇu/) for sperm. We confirmed the phenotype was due to loss of function of SKR by complementation with a transgenic copy. skr/+ plants were transformed with wild type At2g38690 genomic DNA ( gSKR ) by in planta transformation and T1 skr progeny were examined for fertility. The gSKR transgene rescued male sterility confirming the mutant phenotype was due to disruption of At2g38690 ( Fig. 1b , Extended Data Fig. 1d ). Examination of SKR gene expression based on tissue RT-PCR, a proSKR:GFP-GUS reporter, and analysis of publicly available expression profiling data showed that SKR gene expression was specific to reproductive tissues and more specifically to male meiocytes ( Fig. 1d-g ; Extended Data Fig. 2 ). Expression of SKR protein was also meiosis-specific ( Fig. 2c ). Download figure Open in new tab Fig. 2: Staging of skr phenotype and SKR protein expression. (a) Meiotic chromosome development and segregation in wild type (a-j) and corresponding stages in skr (a’-j’). a-d,a’-d’ prophase I; e,e’ metaphase I; f,f’ telophase I; g,g’ metaphase II; h,h’ anaphase II; i,i’ telophase II; j,j’ tetrad. 5846 meiocytes were scored for skr and 2281 for WT. (b) Whole mount DIC images of cleared anthers from successive stage buds; arrowheads indicate degenerating microspores starting in anthers containing early microspores. (c) Anti-GFP staining of male meiosis stages in a proSKR:GFP-gSKR complementing line; asterisks: somatic/tapetal cells; arrowheads: older uninucleate microspores with low GFP signal. Scale bar: (a) 5 µm, (b,c) 20 µm. Early male gametogenesis defects in the shukr mutant Analysis of meiosis and pollen development was performed to identify the stage at which defects arose in skr . Pollen development is divided into two stages: microsporogenesis comprising meiosis together with microspore development, and microgametogenesis or male gametophyte development comprising development of the haploid microspore into pollen 60 . No defects were observed in skr during meiosis based on analysis of chromosome spreads ( Fig. 2a ). Light microscopic examination of whole mount cleared anthers and plastic sections, showed that tetrad formation and release of microspores took place in skr ( Figs. 1h , 2b ; Extended Data Fig. 3 ), however, microspores degenerated shortly after release from tetrads ( Fig. 2b ; Extended Data Fig. 4 ). Newly released wild type microspores were angular in shape ( Fig. 2b ) 61 whereas skr microspores were more rounded and variable in cell wall thickness ( Fig. 2b ), suggesting loss of rigidity and possible defects in cell wall development. Hence sterility in skr appeared to be associated with defects in male gametogenesis between tetrad formation and early gametophyte development. Ultrastructural examination of tetrad and early microgametogenesis stages ( Fig. 3a ) by transmission electron microscopy revealed that a significant proportion of skr tetrads harbored microspores in which the plasma membrane (PM) was retracted from the surrounding callose wall ( Fig. 3b-d ). PM retraction was associated with presence of material between the PM and callose layer likely reflecting unincorporated cell wall precursors or disrupted material arising from disturbance in cell wall development. Hence onset of the mutant phenotype was detected at the tetrad stage. To test for additional defects in skr , we examined expression of the LAT52:GFP reporter that specifically marks the single vegetative cell after pollen mitosis 1 62 . Among skr microspores in which expression of LAT52:GFP took place, we observed two signals in 30% of cases ( Fig. 3e ). Therefore loss of SKR led to at least two distinct defects in early gametogenesis: retraction of the PM with associated disruption of cell wall development, and aberrant expression of an early differentiation marker. Download figure Open in new tab Fig. 3: Ultrastructure and marker expression reveal early gametogenesis defects in skr . (a-c) Ultrastructure of tetrad and early uninucleate microspores showing onset of mutant phenotype at tetrad stage in skr . (a) WT: Normal tetrad and microspore; skr : normal tetrad, degenerating microspore with retracted PM. (b) skr tetrad in which one spore shows PM retraction from callose wall. (c) Magnified image of boxed region from (b) showing PM retraction from callose wall (Cal) and disrupted cell wall material (arrowhead). (d) Quantitation of normal spores and spores showing PM retraction and broken cell wall material (R&B) in tetrads (Fisher’s exact test p < 0.001). (e) Presence of two LAT52:GFP signals in a proportion of skr microspores (Fisher’s exact test p < 0.001). Scale bar (a): Tetrad images 5 µm; microspore images 2 µm; (b) 5 µm; (C) 1 µm. Recessive gene action in the diploid sporophyte would give Mendelian 3:1 segregation of the mutant allele. Gene requirement in the haploid male gametophyte would result in reduced transmission of the mutant allele through the male and a deviation from this ratio. The genetic analysis suggested that SKR acts sporophytically as we did not observe a significant deviation from this ratio ( Fig. 4a ), whereas the mutant phenotype is with respect to the early gametophytic phase. We tested whether SKR also has a gametophytic component to its action by reciprocal crosses of skr/+ to wild type and examining transmission frequency of the mutant skr allele through the gametophyte in comparison to the wild type allele. This test measures fitness of mutant relative to wild type gametes from the same flowers. If there is a functional requirement for SKR gene expression at the haploid stage in the gametophyte, then mutant pollen would show reduced fitness and the mutant allele would be transmitted to the next generation at lower frequency than the wild type allele. The results indicated no significant difference in transmission frequency of the mutant skr allele from that of the wild type ( Fig. 4a ), thus failing to provide evidence for SKR gene action within the gametophyte. Therefore the genetic results together with ultrastructure and marker expression indicated that SKR gene action is in the diploid sporophyte and is required for proper execution of early stages of gametogenesis. Further tests confirmed this finding (see below). Download figure Open in new tab Fig. 4: SHUKR is a gametogenesis inhibitor. (a) Reciprocal crosses comparing transmission of skr and wild type alleles; numbers in brackets are those expected for equal transmission and χ 2 test p-value. (b) Tissue/cell enrichment categories for upregulated genes in skr . (c) Pollen viability in WT and skr plants transgenic for proMSP1:SKRcds or control. Data are pools of 4-10 anthers from 5 independent transgenic lines in each case. Scale bar (c) 100 µm. Meiotic control of male gametophyte gene expression by SHUKR The sporophyte is known to play an important role in formation of the pollen wall and aperture 60 , 63 . Much of the material and machinery for building the outer wall (exine), is synthesized and released by the tapetum, a layer of nurse cells adjacent to the meiocytes 47 , 64 . Examination of SKR gene expression described above indicated that SKR is expressed specifically in male meiocytes. Therefore unlike the sporophytic genes which function in the tapetum, SKR appears to act in meiosis. We determined the localization of SKR protein in the course of meiosis using a complemented line carrying a proSKR:GFP-gSKR gene fusion. SKR protein was detected specifically in male meiocytes from prophase to tetrad stage and extending to early microspores. Subcellular localization of GFP-SKR varied during meiosis, from nuclear enrichment at prophase, changing toward cytoplasmic in late prophase, and back to nuclear at the tetrad stage. Signal intensity was highest in tetrads ( Fig. 2c ). Localization in early microspores shifted from nuclear towards more of cytoplasmic, before declining to an undetected level in later stage uninucleate microspores (UNM). Expression was not detected in tapetal cells ( Fig. 2c ). Therefore SKR protein is mainly present during meiosis and the onset of the skr mutant phenotype corresponds to the tetrad stage at which SKR signal is highest. High SKR expression in tetrads followed by steep decline in free microspores suggests a possible role in repressing gametogenesis or else in ending the sporophytic program before initiation of gametogenesis. To investigate the above possibilities we examined gene expression during meiosis by RNAseq profiling of meiotic anthers (extending upto and not beyond tetrad stage) for skr and wild type. 619 genes were differentially expressed in skr of which 379 genes were upregulated (Supplementary Table 1). We analyzed upregulated genes based on cell and tissue expression in publicly available datasets using the CoNekT platform 65 and found a dramatic enrichment for male gametogenesis-enriched genes 66 , 67 ( Fig. 4b , Supplementary Table 2). 187/379 (49%) of upregulated genes were male gametogenesis-enriched. Genes showing maximal enrichment in UNMs represented 80%, followed by bicellular and tricellular pollen stages (16%). Upregulated genes included DML3 which has been suggested to be an early gametogenesis marker 68 . Increased expression of a large set of gametogenesis-enriched genes in meiotic anthers of skr suggests that SKR negatively regulates expression of gametogenesis genes and represses gametogenesis. SHUKR acts sporophytically and inhibits gametogenesis To test for a role in repression of gametogenesis as well as the possibility that SKR protein acts postmeiotically to promote pollen development, we adopted a misexpression strategy by directing expression of SKR in microspores using the well characterized microspore specific MSP1 promoter 69 . proMSP1:SKR in a skr mutant failed to rescue the mutant phenotype, indicating that SKR expression in the gametophyte is not sufficient to restore function. Furthermore, proMSP1:SKR in wild type in fact caused a reduction in pollen viability ( Fig. 4c ). These results show that expression of SKR during gametogenesis interferes with gametophyte development and argue against the possibility that SKR protein present during postmeiotic stages contributes to pollen development. Therefore SKR acts sporophytically and is an inhibitor of male gametogenesis. SHUKR represses the ubiquitin proteasome system Gene ontology (GO) 70 together with further analysis of individual gene families revealed a striking signature of regulated protein turnover among the 379 genes upregulated in skr RNAs eq. 23% of upregulated genes were associated with ubiquitin-mediated degradation of proteins 71 ( Fig. 5a , Supplementary Table 3; p < 2.0e-33). At the level of molecular function, there was enrichment for ubiquitin protein transferase activity covering the Skp1-Cullin-Fbox (SCF) complex of E3 ubiquitin ligase as well as other components of the ubiquitin protease system (UPS) including RING/U-box and ARIADNE genes which encode E3 ubiquitin ligases 72 , 73 . F-box genes encode the substrate specificity component of SCF and comprised the largest family among upregulated genes in skr . F-box genes in Arabidopsis make up a family of approximately 740 genes 74 , 75 (Supplementary Table 3), several of which show expression during male gametogenesis 76 . 69 F-box genes representing 9% of all F-box genes in Arabidopsis were upregulated in skr (R.F. = 6.6, p < 1.74e-36; Fig. 5a ; Supplementary Table 3). Hence a significant fraction of F-box genes are implicated in pollen development. 75% of upregulated F-box genes were maximally enriched at the UNM stage and 9% at later stages comprising bicellular and tricellular pollen. Among downregulated genes, there was enrichment for stress and hypoxia related genes (Supplementary Table 3). Upregulation of a large number of UPS genes suggested involvement of SKR in general control of protein homeostasis and further experiments were performed to test this hypothesis (see below). We also examined stage-wise expression of a subset of upregulated UPS genes at the cellular level, by generating lines carrying a GFP gene fusion for three different F-box genes ( At3g28330 , At5g02980 and At5g62510 ) and segregating for skr . In each case, we observed F-box:GFP expression in wild type only during postmeiotic development of microspores into pollen, whereas skr showed precocious F-box:GFP expression in meiosis ( Fig. 5b , Extended Data Fig. 5 ). Hence SKR can be seen to act in meiosis to bring about repression of these F-box genes. Taken together, these observations led us to hypothesize that SKR represses onset of male gametogenesis during meiosis through timing of expression of a large set of male gametophyte-preferred F-box and other UPS genes. Download figure Open in new tab Fig. 5: SHUKR regulates the proteasome in pollen development. (a) Gene family categories for upregulated genes in skr . (b) Stagewise expression of an At3g28330:2xnlsGFP transgene during meiosis and in microspores for skr and wildtype. Frames: a,f meiocytes; b,g tetrad; c-e,h microspores; upper half: GFP, lower half: DIC. (c) Suppression of skr male sterility by ski2 . Bar graph shows viable pollen counts in single anthers from 2-4 plants. (d) Downregulation of proteasome genes in skr ski2 . (e) Mean viable pollen counts/anther in independent skr; proSKR:PBE1RNAi and control T1 transgenic plants. (f) Pollen viability increase in skr following proteasome inhibitor treatment in 3 replicate experiments. (g) Model for sporophytic control of pollen development by SHUKR . During meiosis SHUKR establishes a programme that determines timing of expression of F-box and other UPS genes following meiosis. Scale bar: (b) 5 µm; (c) plant 1 cm, anthers 100 µm. Abbreviations: UNM, uninucleate microspore; BCP, bicellular pollen; TCP, tricellular pollen; MP, mature pollen; SGT, sporophyte-gametophyte transition. Control of protein homeostasis by SHUKR We directly investigated the functional basis for the skr phenotype by performing a suppressor screen on skr using EMS mutagenesis of seeds collected from a large number of skr plants followed by screening of M2 plants for improved seed set. We isolated a suppressor of skr which showed elongated siliques and increased pollen viability, whereas skr shows low pollen viability. The suppressor line was backcrossed to the parent skr strain and F1 plants were sterile. F2 seed were collected from multiple F1 plants and grown to give F2 plants which showed recessive Mendelian inheritance for fertility ( Extended Data Fig. 6a ). Whole genome sequencing of bulk segregant fertile and sterile plants from the backcrossed F2 population led to identification of an SNP at the SKI2 locus which created a stop codon after 517 amino acids and showed 100% linkage to the suppressor phenotype ( Fig. 5c , Extended Data Fig. 6b ). SKI2 encodes the RNA helicase subunit of the SKI complex and prevents secondary siRNA production in Arabidopsis 77 . ski2 has also been isolated as a suppressor of overexpression phenotypes in a different context 78 . We tested an independent allele of ski2 by crossing to skr and observed suppression of the skr mutant phenotype, confirming ski2 as a suppressor of skr ( Extended Data Fig. 6c ). We then performed RNAseq on meiotic anthers of the skr ski2 backcrossed suppressor line. Comparison of differentially expressed genes with the skr parent revealed a prominent signature of the proteasome and translation among downregulated genes: 56% of genes encoding subunits of the proteasome and 29% of translation related genes were down-regulated in skr ski2 ( Fig. 5d , Supplementary Table 4). Notably, F-box genes as a class were not downregulated in skr ski2 (R.F.= 0.3; p < 7.7e-19). The striking reduction in transcript levels of the majority of proteasomal genes in skr ski2 strongly supports the hypothesis that the skr phenotype arises from uncoordinated degradation by the proteasome, of regulator(s) of gametogenesis due to inappropriate expression of gametogenesis specific F-box and possibly other ubiquitination genes. Reduction of proteasome activity by downregulation of a major subset of proteasome genes rescues the mutant phenotype. We confirmed involvement of the proteasome in the skr phenotype using two different approaches. First, we performed knockdown of PBE1, a core component of the proteasome 79 in the skr mutant background using pro SKR to direct meiosis-specific expression of PBE1 hairpin RNA and scored for pollen viability in RNAi plants. Knockdown of PBE1 by RNAi has been used in a different context 80 and we used the same region for hairpin RNA construction in our experiments. Second, we treated inflorescences of skr mutant plants with two physiological inhibitors of the proteasome (MG132 and MG115). In both regimes, we observed an increase in formation of viable pollen that was significant for PBE1:RNAi and MG115 ( Fig. 5e,f ; p < 0.05). Taken together, these results clearly establish deregulation of proteasome activity as a major basis for the skr mutant phenotype. The large number of F-box genes showing deregulation during meiosis in skr , most of which normally are preferentially expressed only after meiosis during gametogenesis, highlights the scope of this control. Hence, SKR plays a crucial role in meiosis to repress a proteasomal program that promotes gametogenesis and which comes into play in free microspores following meiosis. SKR therefore sporophytically regulates postmeiotic development of the gametophyte through timing of regulated protein turnover by the proteasome ( Fig. 5g ). Notably, this programme is distinct from the canonical meiotic processes of chromosome reduction and spore formation, which remain unaffected in skr . SHUKR is eudicot specific and rapidly evolving A cDNA of the SKR coding region was amplified by RT-PCR, sequenced, and found to encode a predicted protein of 191 amino acids in agreement with the annotated CDS. Homologs were found only in plants and bore no similarity to any known protein family. SHUKR is therefore the founding member of a new protein family (hereinafter SHUKR). The only alignable region conserved across all SHUKR homologs is always at the C-terminus and this is usually the only globular domain in the protein from Shannon entropy-based compositional analysis. The domain has a central compositionally biased, poorly structured variable region enriched in negatively charged residues. Thus, the SHUKR domain varies in length from 67 to 119 residues. It has a hallmark CxC motif and a dyad of aromatic amino acids (usually Y or F) close to the end of the domain ( Fig. 6a , Supplementary Figure 1). The SHUKR family is present in both great divisions of eudicots: the Superrosids and Superasterids. It is additionally present in Proteales (Nelumbo) and Ranunculales (Macleaya, Papaver), which are the two basal-most branches of eudicots. It is absent in monocots and basal angiosperms. Hence the origin of SHUKR is at the base of eudicots. Download figure Open in new tab Fig. 6: Sequence and evolutionary analysis of the SHUKR domain. (a) Multiple sequence alignment of the SHUKR domain. Lines mark CxC and aromatic dyad. (b) Phylogenetic tree of the SHUKR domain from 119 species. Dark green, Brassicaceae+Cleomaceae; Light green, Asteraceae. (c) Distribution of mean-centered Maximum Likelihood (ML) distance differences. (d) Columnwise Shannon entropy distributions for four plant families. P-value calculated using Kruskal-Wallis test for Brassicaceae and Asteraceae relative to the other two families. To understand evolution of the SHUKR family we used the SHUKR domain of 119 representatives from 85 species for tree construction. Branch length analysis of the tree revealed two lineages displaying elevated evolutionary rates: Brassicaceae+Cleomaceae and Asteraceae ( Fig. 6b ; Extended Data Fig. 7 ). This was further tested using the maximum-likelihood distance matrix computed from the input alignment for the tree. The mean pairwise distance was calculated between each operational taxonomic unit (OTU) and rest of the OTUs, and centered using overall mean distance for the matrix. These mean-centered distances showed a bimodal distribution with the main mode between −1.25 to 0.5, a second mode from 0.5 to 1.5, and a few outliers beyond that ( Fig. 6c ). The second mode corresponds to sequences with higher values of mean-centered distance, indicating their more extensive divergence. Brassicaceae+Cleomaceae (p = 1.1472e-04) and Asteraceae (p = 1.6e-16) were significantly enriched among sequences with mean-centered distance greater than 0.5 indicating they represent atypically fast-evolving groups. We note that the high divergence of the SHUKR family outside the SHUKR domain posed challenges in alignment across the entire coding sequence. Therefore the analysis likely represents a conservative assessment of evolutionary rates within the SHUKR gene family. SHUKR is under positive selection We assessed rapid evolution in particular plant clades by computing column-wise Shannon entropy from an alignment of the SHUKR domain for four plant families with good representation in the Genbank nr database: Brassicaceae, Asteraceae, Fabaceae and Rosaceae. The column-wise entropy was computed using both the 20 amino acid alphabet and a reduced 7 amino acid alphabet that groups together residues as per their shared sidechain chemistry (see Methods). Elevation of entropy in the reduced alphabet indicates changes in sidechain properties along a column and is a proxy for diversifying selection 81 . Entropy plots in both alphabets indicated significantly elevated entropy in Brassicaceae and Asteraceae relative to other families, suggesting action of diversifying selection in these lineages ( Fig. 6d ). At least 30% of species examined had more than one paralog per genome most of which appeared to represent recent lineage-specific duplications as they tended to group together in the tree. However, Asteraceae appears to have experienced a duplication early in their history. Likewise the duplication in Brassicaceae and Cleomaceae predated their divergence. We further assessed diversifying selection at SHUKR within Brassicaceae based on an alignment spanning the length of the gene, using branch-site models 82 that consider proteins as a mosaic of conserved and divergent domains,. The branch-site test results provided further support for existence of positive selection (p = 0.0031) ( Extended Data Table 1 ; Extended Data Fig. 8 ; Supplementary Figure 2), demonstrating that gene duplication has been followed by diversifying selection at SHUKR . Adaptive value for variation in protein turnover during male gametogenesis Plant F-box genes have undergone independent lineage-specific expansions as well as reductions during land plant evolution 83 , 84 . The Brassicaceae family has an expanded set of F-box genes, a majority of which are of short taxonomic scale indicative of more recent emergence 84 . These recent members were significantly enriched among upregulated F-box genes in skr (p < 1.5e-05) ( Fig. 7a,c ; Supplementary Table 5). Orthology analysis of the total set of F-box genes from 10 eudicot species 84 indicated that 58% (37/64) of F-box genes upregulated in skr belonged to orthogroups that included Arabidopsis lyrata but not 8 other eudicot species examined spanning 7 families including Carica papaya (family Caricaceae , order Brassicales ), suggesting that these were likely specific to the Brassicaceae lineage ( Fig. 7b ; Supplementary Table 5). 8% (5/64) of genes belonged to orthogroups having only A. thaliana and not A. lyrata suggesting a more recent origin, and 23% (15/64) had at least one other species but not A. lyrata indicative of recent loss ( Fig. 7b ). We examined the possibility of diversifying selection at the set of skr upregulated genes by testing whether the ratio K a /K s of non-synonymous to synonymous substitutions per site was higher relative to the overall set of F-box genes. Plots of K a /K s gave significantly higher values for the set of F-box genes upregulated in skr relative to the overall set of F-box genes (p = 0.0075; Fig. 7d ). Hence the set of skr upregulated F-box is itself undergoing diversifying selection. Interestingly, male gametes have been proposed as a source for emergence of new genes in both animals and plants 85 – 87 , and pollen-preferred F-box genes also appear to belong to this set. Variation in protein turnover during male gametophyte development can therefore be seen to operate at two levels in evolution. One is through rapid evolution and positive selection in a regulator of UPS genes involved in male gametophyte development. The second is in changes and diversifying selection in components of the UPS system itself, the set of F-box genes that direct substrates for proteasomal degradation in gametogenesis. The large number of F-box genes involved and their distribution across the F-box phylogenetic tree ( Fig. 7c ) suggests the likelihood of levels of multiple proteins being subject to regulation by SKR. Variation in this control provides a plausible route for the sporophyte to influence development and evolution of the gametophyte. These findings lead us to hypothesize that processes that provide for variation in levels of large sets of proteins rather than just a single target during male gametophyte development have adaptive value and have contributed significantly to evolution in Brassicaceae. Download figure Open in new tab Fig. 7: Diversifying selection in SHUKR regulated F-box genes. (a) Enrichment for short taxonomic scale (STS) F-box genes (R.F. = 1.2) and underenrichment for long taxonomic scale (LTS) genes (R.F. = 0.1) among skr upregulated set (p < 1.49e-05). (b) 37 genes were unique to the Brassicaceae lineage of which 5 were found only in Arabidopsis thaliana . 15 genes were present in Arabidopsis thaliana and at least one other species but absent in Arabidopsis lyrata . (c) Phylogenetic tree of Arabidopsis F-box genes showing skr upregulated, LTS, and STS sets. (d) Violin plot of K a /K s for skr upregulated F-box and total F-box genes. Other eudicots: Cucumis sativus , Glycine max , Manihot esculenta , Mimulus guttatus , Populus trichocarpa , Ricinus communis , Vitis vinifera . Abbreviations: Al Arabidopsis lyrata , At Arabidopsis thaliana , Cp Carica papaya . Discussion Extensive functional and gene expression studies of gametophyte development 15 , 18 , 88 have supported the view that the male gametophyte retains control of its development even after evolutionary reduction to just a few cells enclosed within the sporophyte. Experiments in angiosperm species have also shown that isolated microspores can form pollen in culture 52 , 53 , 89 , supporting this view. Here we have demonstrated in Arabidopsis that the sporophyte in fact plays an important role in control of gametogenesis. This direction is by a repressive mechanism involving large scale control of regulated protein turnover in male gametophyte development by a novel gene, SHUKR . SHUKR acts during meiosis and is an inhibitor of gametogenesis ( Fig. 5g ). Our findings in Arabidopsis suggest that significant aspects of the male gametogenesis programme are established in the diploid sporophyte during meiosis but held in a repressed condition until microspores are released. We suggest the term sporophyte directed gametogenesis (SDG) for this phenomenon. Recent emergence of SHUKR at the base of eudicots suggests that SDG arose late in evolution following reduction of the gametophyte. Gametophyte reduction and its close proximity to the sporophyte may have provided conditions necessary for the emergence of SDG. Rapid evolution and diversifying selection at SHUKR and the set of F-box genes under its control points to an adaptive value for variation in protein turnover in male gametophyte evolution. This variation provides a way for the sporophyte to generate diversity in the male gametophytes which face intense selective pressure in the course of transmission and intraspecific competition 90 . The location of this control in the sporophyte is also of interest because it raises the possibility that gametophyte development can be modulated by the sporophyte as a function of its own status and environment. Extended Data Figure Legends Download figure Open in new tab Extended Data Fig. 1: (a) Gene diagram of SKR showing location of insertions: skr-1 (GT_5_101780), skr-2 (CSHL_GT5061), skr-3 (SALKSeq_034497.1). (b) Toluidine blue stained tetrads. (c) Inflorescence stem and Alexander staining showing recessive male sterility of skr. (d) Complementation of skr-3 by a gSKR transgene. Scale bar: 1 cm for inflorescence, 100 µm for anthers. Download figure Open in new tab Extended Data Fig. 2: Profiling of SKR expression in male germline development. TPM values for SKR were collected from the CoNekt database, except for meiocytes. Meiocyte TPM value were taken from (Walker 2018) 21 . UNM – Uninucleate microspore; BCP – Bicellular pollen; TCP – Tricellular pollen; MP – Mature pollen. Download figure Open in new tab Extended Data Fig. 3: Sections of anther development stages. Corresponding stages are shown for wild type (A-D, I-L) and skr (E-H, M-P). Stages are numbered according to (Sanders 1999) 9 . Abbreviations: E epidermis, En endothecium, ML middle layer, T tapetum, Td tetrad, Msp microspore, DM degenerating microspore, PG pollen grain, St stomium, MD microspore debris. Scale bar 20 µm. Download figure Open in new tab Extended Data Fig. 4: Staging of microspore degeneration in skr . Microspore stage and phenotypes are shown together with stage of ovules from the same bud (Schneitz 1995) 10 as a reference for comparison to wild type Download figure Open in new tab Extended Data Fig. 5: Precocious expression of F-box gene:nlsGFP fusions in meiosis in skr . (a) At5g02980. A-C, A’-C’: wild type; D-F, D’-F’: skr; A,A’,D,D’ meiocytes; B,B’,E,E’: tetrad; C,C’,F,F’: uninucleate microspores. (b) At5g62510. A-E, A’-E’: wild type; F-H, F’-H’: skr; A,A’,F,F’ meiocytes; B,B’,G,G’: tetrad; C-E, C’-E’,H,H’: uninucleate microspores. A-H: GFP fluorescence; A’-H’: corresponding DIC. GFP expression in skr is first detected in meiocytes (At5g02980) or tetrad stage At5g62510) whereas in wild type earliest GFP signal was in released microspores. Scale bar: 5 μm Download figure Open in new tab Extended Data Fig. 6: ski2 is a suppressor of skr . (a) Mendelian segregation of the suppressor mutation in the 08-10 skr suppressor line (b) A C>T transition in the ski2 allele (named ski2-7 ) present in the 08-10 skr suppressor line results in a TGA termination codon at position 518. (c) Restoration of fertility and pollen viability in skr-3 by the independent allele ski2-6 . Scale bar: inflorescence 1 cm; anthers 100 μm. Download figure Open in new tab Extended Data Fig. 7: Phylogenetic tree of the SHUKR family from eudicots. 119 representatives from 85 species were used for tree construction using only the SHUKR domain that is shared by all representatives of the family. Genbank ID and species are indicated. Brassicaceae+Cleomaceae (dark green) and Asteraceae (light green) Download figure Open in new tab Extended Data Fig. 8: Phylogenetic tree of Brassicales SHUKR and SHUKR-like ( SKL ) genes used for branch-site test. The SHUKR gene family is represented by a single gene in Carica papaya , whereas Brassicaceae and Cleomaceae experienced a gene duplication event prior to their divergence. CDS were aligned using MAFFT, and RAxML was used to build the phylogenetic tree with 1000 bootstraps. The red branch was selected as foreground for SKR . Branch-site test for positive selection was performed using the CODEML package implemented in EASYCoDEML. Likelihood Ratio Test (LRT) results of Model A vs Model A null (Extended Data Table 1), gave a p-value of 0.0031 for SKR . The tree was unrooted. View this table: View inline View popup Download powerpoint Extended Data Table 1: Branch-site test results for positive selection in the SKR branch in the Brassicaceae. Branch site models postulate three classes of sites: those under purifying selection (0< ω1) in the foreground branch being tested (Yang 2007) 53 . Model A is compared to Model A null in which the third class is set to ω=1. Positive sites are indicated together with posterior probabilities. P-value for likelihood ratio test (LRT) results are calculated from the ξ 2 distribution taking as critical value ξ 2 = 2(LnL MA -LnL MAnull ) and dividing by 2 (Yang 2007) 53 . Methods Plant Material and Growth Conditions Arabidopsis mutant strains used in the study were skr-1 (GT_5_101780), skr-2 (CSHL_GT5061) and skr-3 (SALKSeq_034497.1), and ski2-6 (SALK_122393) 91 . skr-1 and skr-2 alleles are in Ler ecotype and have a Ds insertion in AT2G38690; skr-3 is in Col-0 ecotype. Plants were grown at 22 °C under 16 hours of light and 8 hours of dark in a 1:1:1 proportion of peat, perlite, and vermiculite soaked with 0.5x Murashige Skoog (MS) solution initially followed by irrigation during growth with water. For growth on plates and selection of transformants, seeds were surface sterilized and plated on MS plates supplemented with 2% sucrose and appropriate antibiotics. Coexpression screen for meiotic genes We selected 23 known meiotic genes (DUET/MMD1, MS5/TDM1, DMC1, TAM, SDS, AML5, RAD51, RAD50, ASY1, ZYP1, REC8, SPO11-1, SPO11-2, PHS1, MRE11, MND1, OSD1, NBS1, MSH4, MSH5, HOP2, MER3, PRD1), and each of these genes was used as a query in the Expression Angler program 92 to identify co-expressed genes. For each query, the top 100 hits were selected from each of three data sets, NASCArrays, Bio-analytic resource, and AtGenExpress Plus-Extended tissue compendium. We selected those genes common to three data sets and which appeared at least twice in independent queries. Single copy genes were selected using the Arabidopsis single copy genes list from the University of California, Davis ( http://www.cgpdb.ucdavis.edu/COS_Arabidopsis/ ). We manually selected 29 genes that showed floral stage 9 peak/specific expression corresponding to meiosis stages from AtGenExpress Visualization Tool 93 . We tested one to two insertion alleles for each of the selected genes and for each of these alleles about 20 plants were genotyped by PCR to identify homozygous mutant plants and these were phenotyped by examining silique length and pollen viability by Alexander stain 94 . Plasmid constructs and transgenics For complementation, a genomic fragment of 2220 bp (gSKR) comprising the SKR gene including 3’UTR was amplified from Ler ecotype and cloned into a modified binary vector pZP222 by Gateway cloning (Invitrogen). The binary construct was introduced into Agrobacterium strain AGL1 and transformed into skr-1/+ and skr-3/+ plants by in planta transformation 95 . skr mutant T1 plants were genotyped by PCR and tested for complementation. Plant vectors were ordered from ( https://gatewayvectors.vib.be/index.php/gateway-vectors ). The pro SKR : GFP-gSKR gene fusion construct was made by assembling the SKR promoter, GFP, and SKR genomic coding region plus 3’UTR using three-way Gateway technology in the binary destination vector pB7m34GW,0 96 . The pro SKR:nlsGFP-GUS construct comprised 1170 bp genomic DNA from 690 bp upstream of ATG upto 30 bp of the second exon fused in frame with GFP-GUS in the pBGWFS7 vector. F-box gene nlsGFP fusions were assembled in the pBnRGW_Redseed vector and introduced into plants. Single locus insert transgenic skr/+ lines were made homozygous for the transgene and examined in T4 for GFP fluorescence. The hairpin RNAi construct for PBE1 80 was expressed under control of SKR regulatory elements comprising promoter and 3’UTR and contained the PDK intron from pBm42GWIWG8_1. The construct and a noRNAi control were introduced into skr/+ plants and transgenics that were skr mutant were analysed for pollen viability. The skr-1 mutant allele was used for all studies unless indicated otherwise. proMSP1:SKRcds constructs were made via overlap extension PCR, cloned into pENTR D TOPO vector using NotI and AscI restriction mediated cloning, and further cloned into pZP222 via Gateway cloning. Cytology and imaging For Alexander staining, anthers from unopened buds were dissected out on a slide, placed under a coverslip, and Alexander stain was added from the side to fill the entire coverslip followed by sealing using nail-polish. The slides were incubated at room temperature for 3-4 days and visualized under 10x /20 x objective.Whole anther and ovule clearing was performed as in 97 , anther staging of plastic sections was performed as in 98 and ovule staging as in 99 . Immunostaining for GFP:SKR in meiosis was performed as in 100 . Acid chromosomal spreads were prepared as described in 101 . GFP / DAPI fluorescence was visualized after mounting of meiotic anthers under the coverslip with 30-50 % glycerol / Antifade. ImageJ and Photoshop were used to edit and assemble images. Electron microscopy sample preparation and imaging was done as in 102 and stripe artifacts in the EM images were removed using the method mentioned in 103 . Suppressor screening and analysis skr-1 mutant seeds (M0) were bulked from multiple plants and 0.3 % EMS solution was used to mutagenise the M0 seeds for 5 hours followed by extensive washing with water. Seeds from 934 M1 plants were collected individually and M2 families were screened for fertility restoration. One suppressor line 08-10 was identified and backcrossed to skr mutants to get BC1 F1 seeds. Seeds from BC1 F1 plants were collected and bulk segregant analysis on BC1 F2 plants was performed on fertile and sterile pools. Genomic DNA was isolated from the fertile and sterile pools (50 plants each) and used for whole genome sequencing. To map the suppressor, the parental un-mutagenised skr genome was sequenced and skr specific reference genome was made by replacing the SNPs of skr to Ler-0 genome 104 sequence using the bedtools – consensus 105 . Variants were called for the fertile and sterile bulk reads from the skr reference genome using freebayes 106 and indels were removed. SNPindex was calculated using the R package QTLSeqr 107 for the fertile bulk and sterile bulk. SNPs with a deltaSNP of fertile to sterile score greater than 0.5 were analysed for genic perturbations. The 08-10 line had an in frame stop codon in the SKI2 gene, at amino acid position 518. The suppressor phenotype was confirmed by generating the double mutant using the independent alleles ski2-6 and skr-3. The ski2 allele in line 08-10 was named ski2-7 . Expression profiling RNA from meiotic stage anthers was isolated using Qiagen kits for skr-1, Ler and skr-1 ski2-7 . RNA sequencing was performed on Illumina platforms following the manufacturer’s instructions. Differentially expressed genes were found using DESeq2 keeping a FDR adjusted P value < 0.05 108 . Validation for eight upregulated F-box genes was performed by RT-qPCR (Supplementary Figure 3) which confirmed the RNAseq results. The tissue-specific expression analysis of deregulated genes was performed by downloading TPM values for each gene from CoNekT ( https://evorepro.sbs.ntu.edu.sg ) database 65 . Meiocyte RNAseq TPM value 109 was combined with the CoNekT dataset. The tissue enrichment score was calculated by row normalization using the largest TPM score. Enrichment score of 1, in a tissue/cell type was taken to represent maximal expression in that type. The number of non-overlapping genes with enrichment score 1, was calculated and plotted for meiocytes, UNM, BCP, TCP/MP and others. Proteasome inhibitor treatment MG132 and MG115 stocks at 20 mM concentration were made in DMSO. Inhibitor solutions were used at 100 µM concentration 110 by dissolving in half strength MS where 0.5 % DMSO was present. Control solution was 0.5 % DMSO in half strength MS. Sections of freshly cut skr inflorescence stems were placed in 200 µl inhibitor or control solution in Eppendorf tubes for 3 hrs, followed by 60 hrs of recovery in half strength MS solution. Anthers were dissected and viable pollens were identified by Alexander staining. At least 100 anthers were analysed per sample (treatment and control) per experiment. Number of anthers with 10 or more viable pollen were counted per 100 anthers for treatment and control and the ratio was calculated. The experiment was conducted blind and repeated thrice. The ratio paired t test (two-tailed) on the logarithm of ratios was used to calculate p values (GraphPad). Sequence analysis Sequence searches to detect homologs of the SHUKR domain were conducted using the PSI-BLAST (RRID:SCR_001010) 111 and JACKHMMER (RRID:SCR_005305) 112 programs with profile-inclusion threshold of expect (e)-value at 0.01 against the non-redundant database of National Center for Biotechnology Information (NCBI) clustered down to 50%. Clustering of proteins based on bit score density and length of aligned sequence was performed using the MMseqs program. Remote homology searches were performed using profile-profile comparisons with HHpred program (RRID:SCR_010276) 113 against profile libraries from the PFAM (RRID: SCR_004726) 114 and PDB (RRID:SCR_012820) 115 databases. Multiple sequence alignments were built using Mafft program with maxiterate parameter set to 3000 followed by manual adjustments based on secondary structure prediction. Secondary structures were predicted using the JPred (RRID:SCR_016504) 116 and RoseTTaFold 117 programs. The globular domains in the protein were objectively identified using Shannon entropy-based compositional analysis with the SEG program with a window size of 45, low-cut of 3.4, high-cut of 3.75, and minimal length of the high-complexity segment of 15 residues. The analysis of the Shannon entropy (H j ) for a given multiple sequence alignment was performed using the equation: P is the fraction of residues of amino acid type i and M is the number of amino acid types. The Shannon entropy for the jth position in the alignment can range from 0 (only one residue at that position) to 4.32 (all 20 residues equally represented at that position). The reduced alphabet was created by the following grouping: h= L, I, M, V, A; a= F, Y, W; b=K, R, H; n=E, D; p=Q, N; o=T, C, S; t=P, G, d=-(gap). Phylogenetic analysis SKR and SKL coding sequences of different species were extracted from Genbank, Phytozome, and CoGeBLAST 118 , 119 (Supplementary File 1). Genomic fragments were annotated to CDS by the online tool MEGANTE 120 . For the branch site analysis, MAFFT was used to build the alignment and RAxML was used to build a phylogenetic tree with 1000 bootstraps 121 , 122 . Branch site model tests of the CODEML package 82 were performed as implemented in EasyCoDEML 123 by selecting the branch of choice. The tree was unrooted. Protein phylogenetic analysis was performed using the maximum likelihood method implemented in the IQtree program (RRID: SCR_017254) 124 under multiple parameter regimes using: 1) the Q.plant substitution matrix derived from alignments specific to plant nuclear genes and 1 invariant site category with 8 gamma distributed sites; 2) the LG substitution matrix with 1 invariant site category with 8 gamma distributed sites or 3) with a 20-profile mixture model. Bootstrap values were calculated using the Shimodaira-Hasegawa-like approximate likelihood ratio (SH-aLRT) 125 . F-box gene sequences of 710 Arabidopsis genes used for ortholog analysis were obtained from Ref. 84 of which 64/69 were common to skr upregulated. Classification of LTS and STS genes was according to Ref. 84. Identification of orthologs was done using Proteinortho 126 . Funding This work was supported by a CSIR Network Project Grant MLP0120 and a Department of Biotechnology Centre of Excellence Grant BT/PR21305/COE/34/44/2016 to IS. SP and PS were supported by CSIR Fellowships. AR was supported by a Department of Biotechnology Research Associate Fellowship. IS acknowledges a JC Bose fellowship from the Department of Science and Technology and a Senior Scientist fellowship from the Indian National Science Academy. Author contributions PS, SP, JND, JD and IS conceived and planned experiments. SP, PS, AR, JND, VS, HA, AS, and IS performed experiments. SP, PS, AR, AS, CK, HG, JD, and IS provided analysis of the experimental results. LA contributed the evolutionary analysis of Figure 6 . IS and PS wrote the manuscript with contributions from LA and JD. Competing interests Authors declare no competing interests. Additional information Supplementary information is available for this paper. Materials and correspondence Correspondence and requests for materials to be addressed to IS. Sequencing data will be deposited at the European Nucleotide Archive. Supplementary information Supplementary Table 1: Differentially expressed gene lists. Supplementary Table 2: Male gametogenesis enrichment analysis of skr upregulated genes. Supplementary Table 3: Analysis of genes differentially expressed in skr . Supplementary Table 4: Differentially expressed translation-related genes in skr ski2 . Supplementary Table 5: Enrichment for short taxonomic scale (STS) F-box genes upregulated in skr . Supplementary Table 6: List of primers Supplementary Figure 1: Multiple sequence alignment of the SHUKR domain. Supplementary Figure 2: Multiple sequence alignment of SKR protein sequences used for branch-site analysis. Supplementary Figure 3: Validation for eight skr upregulated F-box genes by RT-qPCR. Supplementary File 1: Sequences of SHUKR family genes used in branch-site analysis. Acknowledgments We thank David Twell for kindly providing the LAT52:GFP reporter line, ABRC and CSHL for seeds. Kathakali Bannerjee and Manoj Kamble for technical support, Mekala Sai Kiran for maintenance of plants, the NGS and Advanced Microscopy facilities at CCMB, and Priyanka Pant for help in illustrations. We are grateful to Elliot Meyerowitz, Utpal Nath, and Manjula Reddy for valuable comments on the manuscript. Footnotes ↵ ‡ databaum GmbH; Holsteinischer Kamp 80, 22081 Hamburg, Germany. ↵ § Seri-Biotech Research Laboratory, Central Silk Board; Bangalore-560035, India. ↵ ¶ Department of Botany, Banaras Hindu University; Varanasi 221005, India. ↵ # University of Texas at Austin; Texas, USA. ↵ †† Deceased One line in the abstract was missed and updated. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1164421 References 1. ↵ Graham , L. E . The Origin of the Life Cycle of Land Plants: A simple modification in the life cycle of an extinct green alga is the likely origin of the first land plants . American Scientist 73 , 178 – 186 ( 1985 ). OpenUrl GeoRef Web of Science 2. ↵ Kenrick , P . Alternation of Generations in Land Plants: New Phylogenetic and Palaeobotanical Evidence . Biological Reviews 69 , 293 – 330 ( 1994 ). OpenUrl CrossRef 3. ↵ Niklas , K. J. & Kutschera , U . The evolution of the land plant life cycle . New Phytologist 185 , 27 – 41 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ Graham , L. E . Origin of Land Plants . Wiley.com https://www.wiley.com/en-us/Origin+of+Land+Plants-p-9780471615279 ( 1993 ). 5. ↵ Delwiche , C. F. & Cooper , E. D . The Evolutionary Origin of a Terrestrial Flora . Curr Biol 25 , R899 – 910 ( 2015 ). OpenUrl CrossRef PubMed 6. ↵ Kenrick , P. & Crane , P. R . The origin and early evolution of plants on land . Nature 389 , 33 – 39 ( 1997 ). OpenUrl CrossRef GeoRef Web of Science 7. ↵ Graham , L. E. , Cook , M. E. & Busse , J. S . The origin of plants: Body plan changes contributing to a major evolutionary radiation . Proceedings of the National Academy of Sciences 97 , 4535 – 4540 ( 2000 ). OpenUrl FREE Full Text 8. ↵ Ma , H. & Sundaresan , V. Chapter Thirteen - Development of Flowering Plant Gametophytes. in Current Topics in Developmental Biology (ed. Timmermans, M. C. P.) vol. 91 379 – 412 (Academic Press, 2010 ). 9. ↵ Harrison , C. J. & Morris , J. L . The origin and early evolution of vascular plant shoots and leaves . Philos Trans R Soc Lond B Biol Sci 373 , 20160496 ( 2018 ). 10. ↵ Sakakibara , K. et al. KNOX2 genes regulate the haploid-to-diploid morphological transition in land plants . Science 339 , 1067 – 1070 ( 2013 ). OpenUrl Abstract / FREE Full Text 11. ↵ Mosquna , A. et al. Regulation of stem cell maintenance by the Polycomb protein FIE has been conserved during land plant evolution . Development 136 , 2433 – 2444 ( 2009 ). OpenUrl Abstract / FREE Full Text 12. ↵ Okano , Y. et al. A polycomb repressive complex 2 gene regulates apogamy and gives evolutionary insights into early land plant evolution . Proc Natl Acad Sci U S A 106 , 16321 – 16326 ( 2009 ). OpenUrl Abstract / FREE Full Text 13. ↵ Strome , S. & Lehmann , R . Germ versus soma decisions: lessons from flies and worms . Science 316 , 392 – 393 ( 2007 ). OpenUrl Abstract / FREE Full Text 14. ↵ Hayashi , K. & Surani , M. A . Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro . Development 136 , 3549 – 3556 ( 2009 ). OpenUrl Abstract / FREE Full Text 15. ↵ Berger , F. & Twell , D. Germline Specification and Function in Plants . Annual Review of Plant Biology 62 , 461 – 484 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 16. ↵ Hisanaga , T. et al. Building new insights in plant gametogenesis from an evolutionary perspective . Nat Plants 5 , 663 – 669 ( 2019 ). OpenUrl CrossRef PubMed 17. ↵ Grossniklaus , U . Plant germline development: a tale of cross-talk, signaling, and cellular interactions . Sex Plant Reprod 24 , 91 – 95 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 18. ↵ Twell , D . Male gametogenesis and germline specification in flowering plants . Sex Plant Reprod 24 , 149 – 160 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 19. ↵ Yamaoka , S. et al. Generative Cell Specification Requires Transcription Factors Evolutionarily Conserved in Land Plants . Curr Biol 28 , 479 – 486 .e5 ( 2018 ). OpenUrl CrossRef PubMed 20. ↵ Saito , M. et al. A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes . Curr Biol 33 , 4980 – 4987 .e6 ( 2023 ). OpenUrl CrossRef PubMed 21. ↵ Borg , M. et al. The R2R3 MYB Transcription Factor DUO1 Activates a Male Germline-Specific Regulon Essential for Sperm Cell Differentiation in Arabidopsis . The Plant Cell 23 , 534 – 549 ( 2011 ). OpenUrl Abstract / FREE Full Text 22. ↵ Higo , A. et al. Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants . Nat Commun 9 , 5283 ( 2018 ). OpenUrl CrossRef PubMed 23. ↵ Kasahara , R. D. , Portereiko , M. F. , Sandaklie-Nikolova , L. , Rabiger , D. S. & Drews , G. N . MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis . Plant Cell 17 , 2981 – 2992 ( 2005 ). OpenUrl Abstract / FREE Full Text 24. Gross-Hardt , R. et al. LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis . PLoS Biol 5 , e47 ( 2007 ). OpenUrl CrossRef PubMed 25. Moll , C. et al. CLO/GFA1 and ATO are novel regulators of gametic cell fate in plants . Plant J 56 , 913 – 921 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 26. Rabiger , D. S. & Drews , G. N . MYB64 and MYB119 Are Required for Cellularization and Differentiation during Female Gametogenesis in Arabidopsis thaliana . PLOS Genetics 9 , e1003783 ( 2013 ). OpenUrl CrossRef 27. Yuan , L. et al. The CKI1 Histidine Kinase Specifies the Female Gametic Precursor of the Endosperm . Developmental Cell 37 , 34 – 46 ( 2016 ). OpenUrl CrossRef PubMed 28. Portereiko , M. F. et al. AGL80 Is Required for Central Cell and Endosperm Development in Arabidopsis . The Plant Cell 18 , 1862 – 1872 ( 2006 ). OpenUrl Abstract / FREE Full Text 29. ↵ Bemer , M. , Wolters-Arts , M. , Grossniklaus , U. & Angenent , G. C . The MADS Domain Protein DIANA Acts Together with AGAMOUS-LIKE80 to Specify the Central Cell in Arabidopsis Ovules . The Plant Cell 20 , 2088 – 2101 ( 2008 ). OpenUrl Abstract / FREE Full Text 30. ↵ Bateman , R. M. & DiMICHELE , W. A . Heterospory: The Most Iterative Key Innovation in the Evolutionary History of the Plant Kingdom . Biological Reviews 69 , 345 – 417 ( 1994 ). OpenUrl CrossRef Web of Science 31. ↵ Feng , X. , Zilberman , D. & Dickinson , H . A conversation across generations: soma-germ cell crosstalk in plants . Dev Cell 24 , 215 – 225 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 32. Schmidt , A. , Schmid , M. W. & Grossniklaus , U . Plant germline formation: common concepts and developmental flexibility in sexual and asexual reproduction . Development 142 , 229 – 241 ( 2015 ). OpenUrl Abstract / FREE Full Text 33. ↵ Petrella , R. , Cucinotta , M. , Mendes , M. A. , Underwood , C. J. & Colombo , L . The emerging role of small RNAs in ovule development, a kind of magic . Plant Reprod 34 , 335 – 351 ( 2021 ). OpenUrl CrossRef PubMed 34. ↵ Yang , W. C. , Ye , D. , Xu , J. & Sundaresan , V . The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein . Genes Dev 13 , 2108 – 2117 ( 1999 ). OpenUrl Abstract / FREE Full Text 35. Schiefthaler , U. et al. Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana . Proc Natl Acad Sci U S A 96 , 11664 – 11669 ( 1999 ). OpenUrl Abstract / FREE Full Text 36. ↵ Mendes , M. A. et al. The RNA-dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in Arabidopsis . Development 147 , dev194274 ( 2020 ). OpenUrl Abstract / FREE Full Text 37. ↵ Zhao , D.-Z. , Wang , G.-F. , Speal , B. & Ma , H . The excess microsporocytes1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther . Genes Dev 16 , 2021 – 2031 ( 2002 ). OpenUrl Abstract / FREE Full Text 38. Canales , C. , Bhatt , A. M. , Scott , R. & Dickinson , H . EXS, a putative LRR receptor kinase, regulates male germline cell number and tapetal identity and promotes seed development in Arabidopsis . Curr Biol 12 , 1718 – 1727 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 39. ↵ Jia , G. , Liu , X. , Owen , H. A. & Zhao , D . Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase . Proc Natl Acad Sci U S A 105 , 2220 – 2225 ( 2008 ). OpenUrl Abstract / FREE Full Text 40. ↵ Nonomura , K.-I. et al. A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice . Plant Cell 19 , 2583 – 2594 ( 2007 ). OpenUrl Abstract / FREE Full Text 41. ↵ Singh , M. et al. Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein . Plant Cell 23 , 443 – 458 ( 2011 ). OpenUrl Abstract / FREE Full Text 42. ↵ Su , Z. et al. The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module . Curr Biol 27 , 1597 – 1609 .e2 ( 2017 ). OpenUrl CrossRef PubMed 43. ↵ Martínez , G. , Panda , K. , Köhler , C. & Slotkin , R. K . Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell . Nat Plants 2 , 16030 ( 2016 ). 44. ↵ Pagnussat , G. C. , Alandete-Saez , M. , Bowman , J. L. & Sundaresan , V . Auxin-dependent patterning and gamete specification in the Arabidopsis female gametophyte . Science 324 , 1684 – 1689 ( 2009 ). OpenUrl Abstract / FREE Full Text 45. ↵ Panoli , A. et al. Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions, Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana . PLoS One 10 , e0126164 ( 2015 ). OpenUrl CrossRef PubMed 46. ↵ Gómez , J. F. , Talle , B. & Wilson , Z. A . Anther and pollen development: A conserved developmental pathway . Journal of Integrative Plant Biology 57 , 876 – 891 ( 2015 ). OpenUrl CrossRef PubMed 47. ↵ Ariizumi , T. & Toriyama , K . Genetic regulation of sporopollenin synthesis and pollen exine development . Annu Rev Plant Biol 62 , 437 – 460 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 48. ↵ Wilson , Z. A. , Morroll , S. M. , Dawson , J. , Swarup , R. & Tighe , P. J . The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors . Plant J 28 , 27 – 39 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 49. Sorensen , A.-M. et al. The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor . Plant J 33 , 413 – 423 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Zhang , W. et al. Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor . Development 133 , 3085 – 3095 ( 2006 ). OpenUrl Abstract / FREE Full Text 51. ↵ Long , J. et al. Nurse cell--derived small RNAs define paternal epigenetic inheritance in Arabidopsis . Science 373 , eabh0556 ( 2021 ). OpenUrl Abstract / FREE Full Text 52. ↵ Tanaka , I. & Ito , M . Induction of typical cell division in isolated microspores of Lilium longiflorum and Tulipa gesneriana . Plant Science Letters 17 , 279 – 285 ( 1980 ). OpenUrl CrossRef 53. ↵ Moreno , R. M. , Macke , F. , Alwen , A. & Heberle-Bors , E . In-situ seed production after pollination with in-vitro-matured, isolated pollen . Planta 176 , 145 – 148 ( 1988 ). OpenUrl CrossRef PubMed 54. ↵ Chaudhury , A. M . Nuclear Genes Controlling Male Fertility . Plant Cell 5 , 1277 – 1283 ( 1993 ). OpenUrl FREE Full Text 55. ↵ Mercier , R. & Grelon , M . Meiosis in plants: ten years of gene discovery . Cytogenet Genome Res 120 , 281 – 290 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 56. ↵ Blackmore , S. , Wortley , A. H. , Skvarla , J. J. & Rowley , J. R . Pollen wall development in flowering plants . New Phytol 174 , 483 – 498 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 57. Scott , R. J. , Spielman , M. & Dickinson , H. G. Stamen structure and function . Plant Cell 16 Suppl, S46 – 60 ( 2004 ). OpenUrl FREE Full Text 58. Bencivenga , S. , Colombo , L. & Masiero , S . Cross talk between the sporophyte and the megagametophyte during ovule development . Sex Plant Reprod 24 , 113 – 121 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 59. ↵ Figueiredo , D. D. & Köhler , C . Bridging the generation gap: communication between maternal sporophyte, female gametophyte and fertilization products . Curr Opin Plant Biol 29 , 16 – 20 ( 2016 ). OpenUrl CrossRef PubMed 60. ↵ McCormick , S . Male Gametophyte Development . Plant Cell 5 , 1265 – 1275 ( 1993 ). OpenUrl FREE Full Text 61. ↵ Owen , H. A. & Makaroff , C. A . Ultrastructure of microsporogenesis and microgametogenesis inArabidopsis thaliana (L .) Heynh. ecotype Wassilewskija (Brassicaceae). Protoplasma 185 , 7 – 21 ( 1995 ). OpenUrl 62. ↵ Twell , D. Use of a nuclear-targeted β-glucuronidase fusion protein to demonstrate vegetative cell-specific gene expression in developing pollen . The Plant Journal https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-313X.1992.00887.x ( 1992 ). 63. ↵ Dobritsa , A. A. & Coerper , D . The novel plant protein INAPERTURATE POLLEN1 marks distinct cellular domains and controls formation of apertures in the Arabidopsis pollen exine . Plant Cell 24 , 4452 – 4464 ( 2012 ). OpenUrl Abstract / FREE Full Text 64. ↵ Ma , H . Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants . Annu Rev Plant Biol 56 , 393 – 434 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 65. ↵ Proost , S. & Mutwil , M . CoNekT: an open-source framework for comparative genomic and transcriptomic network analyses . Nucleic Acids Res 46 , W133 – W140 ( 2018 ). OpenUrl CrossRef PubMed 66. ↵ Honys , D. & Twell , D . Comparative analysis of the Arabidopsis pollen transcriptome . Plant Physiol 132 , 640 – 652 ( 2003 ). OpenUrl Abstract / FREE Full Text 67. ↵ Pina , C. , Pinto , F. , Feijó , J. A. & Becker , J. D . Gene Family Analysis of the Arabidopsis Pollen Transcriptome Reveals Biological Implications for Cell Growth, Division Control, and Gene Expression Regulation . Plant Physiol 138 , 744 – 756 ( 2005 ). OpenUrl Abstract / FREE Full Text 68. ↵ Ichino , L. et al. Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen . Cell Rep 41 , 111699 ( 2022 ). OpenUrl CrossRef PubMed 69. ↵ Honys , D. et al. Identification of microspore-active promoters that allow targeted manipulation of gene expression at early stages of microgametogenesis in Arabidopsis . BMC Plant Biol 6 , 31 ( 2006 ). 70. ↵ Raudvere , U. et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update) . Nucleic Acids Res 47 , W191 – W198 ( 2019 ). OpenUrl CrossRef PubMed 71. ↵ Moon , J. , Parry , G. & Estelle , M . The ubiquitin-proteasome pathway and plant development . Plant Cell 16 , 3181 – 3195 ( 2004 ). OpenUrl FREE Full Text 72. ↵ Smalle , J. & Vierstra , R. D . The ubiquitin 26S proteasome proteolytic pathway . Annu Rev Plant Biol 55 , 555 – 590 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 73. ↵ Mladek , C. , Guger , K. & Hauser , M.-T . Identification and characterization of the ARIADNE gene family in Arabidopsis . A group of putative E3 ligases. Plant Physiol 131 , 27 – 40 ( 2003 ). OpenUrl 74. ↵ Gagne , J. M. , Downes , B. P. , Shiu , S.-H. , Durski , A. M. & Vierstra , R. D . The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis . Proc Natl Acad Sci U S A 99 , 11519 – 11524 ( 2002 ). OpenUrl Abstract / FREE Full Text 75. ↵ Kuroda , H. et al. Classification and expression analysis of Arabidopsis F-box-containing protein genes . Plant Cell Physiol 43 , 1073 – 1085 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 76. ↵ Ikram , S. et al. Functional redundancy and/or ongoing pseudogenization among F-box protein genes expressed in Arabidopsis male gametophyte . Plant Reprod 27 , 95 – 107 ( 2014 ). OpenUrl CrossRef PubMed 77. ↵ Branscheid , A. et al. SKI2 mediates degradation of RISC 5’-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis . Nucleic Acids Res 43 , 10975 – 10988 ( 2015 ). OpenUrl CrossRef PubMed 78. ↵ Zhang , X. et al. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis . Science 348 , 120 – 123 ( 2015 ). OpenUrl Abstract / FREE Full Text 79. ↵ Fu , H. , Doelling , J. H. , Arendt , C. S. , Hochstrasser , M. & Vierstra , R. D . Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana . Genetics 149 , 677 – 692 ( 1998 ). OpenUrl Abstract / FREE Full Text 80. ↵ Hatsugai , N. et al. A novel membrane fusion-mediated plant immunity against bacterial pathogens . Genes Dev 23 , 2496 – 2506 ( 2009 ). OpenUrl Abstract / FREE Full Text 81. ↵ Krishnan , A. , Iyer , L. M. , Holland , S. J. , Boehm , T. & Aravind , L . Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity . Proc Natl Acad Sci U S A 115 , E3201 – E3210 ( 2018 ). OpenUrl Abstract / FREE Full Text 82. ↵ Yang , Z . PAML 4: phylogenetic analysis by maximum likelihood . Mol Biol Evol 24 , 1586 – 1591 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 83. ↵ Xu , G. , Ma , H. , Nei , M. & Kong , H . Evolution of F-box genes in plants: Different modes of sequence divergence and their relationships with functional diversification . Proc Natl Acad Sci U S A 106 , 835 – 840 ( 2009 ). OpenUrl Abstract / FREE Full Text 84. ↵ Hua , Z. , Zou , C. , Shiu , S.-H. & Vierstra , R. D . Phylogenetic Comparison of F-Box (FBX) Gene Superfamily within the Plant Kingdom Reveals Divergent Evolutionary Histories Indicative of Genomic Drift . PLOS ONE 6 , e16219 ( 2011 ). OpenUrl CrossRef PubMed 85. ↵ Kaessmann , H . Origins, evolution, and phenotypic impact of new genes . Genome Res 20 , 1313 – 1326 ( 2010 ). OpenUrl Abstract / FREE Full Text 86. Wu , D.-D. et al. “Out of Pollen” Hypothesis for Origin of New Genes in Flowering Plants: Study from Arabidopsis thaliana . Genome Biology and Evolution 6 , 2822 – 2829 ( 2014 ). OpenUrl CrossRef PubMed 87. ↵ Cui , X. et al. Young Genes out of the Male: An Insight from Evolutionary Age Analysis of the Pollen Transcriptome . Mol Plant 8 , 935 – 945 ( 2015 ). OpenUrl CrossRef PubMed 88. ↵ Nelms , B. & Walbot , V . Gametophyte genome activation occurs at pollen mitosis I in maize . Science 375 , 424 – 429 ( 2022 ). OpenUrl CrossRef PubMed 89. ↵ Barinova , I. et al. Antirrhinum majus microspore maturation and transient transformation in vitro . J Exp Bot 53 , 1119 – 1129 ( 2002 ). OpenUrl CrossRef PubMed 90. ↵ Delph , L. F . Pollen competition is the mechanism underlying a variety of evolutionary phenomena in dioecious plants . New Phytol 224 , 1075 – 1079 ( 2019 ). OpenUrl CrossRef PubMed 91. ↵ Zhao , L. & Kunst , L . SUPERKILLER Complex Components Are Required for the RNA Exosome-Mediated Control of Cuticular Wax Biosynthesis in Arabidopsis Inflorescence Stems . Plant Physiol 171 , 960 – 973 ( 2016 ). OpenUrl Abstract / FREE Full Text 92. ↵ Toufighi , K. , Brady , S. M. , Austin , R. , Ly , E. & Provart , N. J . The Botany Array Resource: e-Northerns, Expression Angling, and promoter analyses . Plant J 43 , 153 – 163 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 93. ↵ Schmid , M. et al. A gene expression map of Arabidopsis thaliana development . Nat Genet 37 , 501 – 506 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 94. ↵ Alexander , M. P . Differential staining of aborted and nonaborted pollen . Stain Technol 44 , 117 – 122 ( 1969 ). OpenUrl CrossRef PubMed Web of Science 95. ↵ Clough , S. J. & Bent , A. F . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J 16 , 735 – 743 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 96. ↵ Karimi , M. , Depicker , A. & Hilson , P . Recombinational cloning with plant gateway vectors . Plant Physiol 145 , 1144 – 1154 ( 2007 ). OpenUrl FREE Full Text 97. ↵ Reddy , T. V. , Kaur , J. , Agashe , B. , Sundaresan , V. & Siddiqi , I . The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein . Development 130 , 5975 – 5987 ( 2003 ). OpenUrl Abstract / FREE Full Text 98. ↵ Sanders , P. M. et al. Anther developmental defects in Arabidopsis thaliana male-sterile mutants . Sex Plant Reprod 11 , 297 – 322 ( 1999 ). OpenUrl CrossRef Web of Science 99. ↵ Schneitz , K. , Hülskamp , M. & Pruitt , R. E . Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue . The Plant Journal 7 , 731 – 749 ( 1995 ). OpenUrl CrossRef Web of Science 100. ↵ Andreuzza , S. , Nishal , B. , Singh , A. & Siddiqi , I . The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis . PLoS Genet 11 , e1005396 ( 2015 ). OpenUrl CrossRef PubMed 101. ↵ Singh , D. K. , Spillane , C. & Siddiqi , I . PATRONUS1 is expressed in meiotic prophase I to regulate centromeric cohesion in Arabidopsis and shows synthetic lethality with OSD1 . BMC Plant Biol 15 , 201 ( 2015 ). 102. ↵ Siddiqi , I. , Ganesh , G. , Grossniklaus , U. & Subbiah , V . The dyad gene is required for progression through female meiosis in Arabidopsis . Development 127 , 197 – 207 ( 2000 ). OpenUrl Abstract 103. ↵ Schwartz , J. et al. Removing Stripes, Scratches, and Curtaining with Nonrecoverable Compressed Sensing . Microscopy and Microanalysis 25 , 705 – 710 ( 2019 ). OpenUrl CrossRef 104. ↵ Jiao , W.-B. & Schneeberger , K . Chromosome-level assemblies of multiple Arabidopsis genomes reveal hotspots of rearrangements with altered evolutionary dynamics . Nat Commun 11 , 989 ( 2020 ). 105. ↵ Quinlan , A. R . BEDTools: The Swiss-Army Tool for Genome Feature Analysis . Curr Protoc Bioinformatics 47 , 11.12. 1 – 34 ( 2014 ). OpenUrl CrossRef PubMed 106. ↵ Garrison , E. & Marth , G. Haplotype-based variant detection from short-read sequencing . Preprint at doi: 10.48550/arXiv.1207.3907 ( 2012 ). OpenUrl CrossRef 107. ↵ Mansfeld , B. N. & Grumet , R . QTLseqr: An R Package for Bulk Segregant Analysis with Next-Generation Sequencing . Plant Genome 11 , ( 2018 ). 108. ↵ Love , M. I. , Huber , W. & Anders , S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol 15 , 550 ( 2014 ). 109. ↵ Walker , J. et al. Lineage-specific DNA methylation regulates Arabidopsis sexual development . Nat Genet 50 , 130 – 137 ( 2018 ). OpenUrl CrossRef PubMed 110. ↵ Xu , D. et al. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation . Proc Natl Acad Sci U S A 113 , 7655 – 7660 ( 2016 ). OpenUrl Abstract / FREE Full Text 111. ↵ Altschul , S . Gapped BLAST and PSI-BLAST: a new generation of protein database search programs . Nucleic Acids Research 25 , 3389 – 3402 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 112. ↵ Potter , S. C. et al. HMMER web server: 2018 update . Nucleic Acids Res 46 , W200 – W204 ( 2018 ). OpenUrl CrossRef PubMed 113. ↵ Zimmermann , L. et al. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core . J Mol Biol 430 , 2237 – 2243 ( 2018 ). OpenUrl CrossRef PubMed 114. ↵ Mistry , J. et al. Pfam: The protein families database in 2021 . Nucleic Acids Res 49 , D412 – D419 ( 2021 ). OpenUrl CrossRef PubMed 115. ↵ Berman , H. M. et al. The Protein Data Bank . Nucleic Acids Res 28 , 235 – 242 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 116. ↵ Drozdetskiy , A. , Cole , C. , Procter , J. & Barton , G. J . JPred4: a protein secondary structure prediction server . Nucleic Acids Res 43 , W389 – 394 ( 2015 ). OpenUrl CrossRef PubMed 117. ↵ Baek , M. et al. Accurate prediction of protein structures and interactions using a three-track neural network . Science 373 , 871 – 876 ( 2021 ). OpenUrl Abstract / FREE Full Text 118. ↵ Goodstein , D. M. et al. Phytozome: a comparative platform for green plant genomics . Nucleic Acids Res 40 , D1178 – 1186 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 119. ↵ Lyons , E. et al. Finding and comparing syntenic regions among Arabidopsis and the outgroups papaya, poplar, and grape: CoGe with rosids . Plant Physiol 148 , 1772 – 1781 ( 2008 ). OpenUrl Abstract / FREE Full Text 120. ↵ Numa , H. & Itoh , T . MEGANTE: A Web-Based System for Integrated Plant Genome Annotation . Plant Cell Physiol 55 , e2 ( 2014 ). OpenUrl CrossRef PubMed 121. ↵ Katoh , K. & Standley , D. M . MAFFT multiple sequence alignment software version 7: improvements in performance and usability . Mol Biol Evol 30 , 772 – 780 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 122. ↵ Stamatakis , A . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies . Bioinformatics 30 , 1312 – 1313 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 123. ↵ Gao , F. et al. EasyCodeML: A visual tool for analysis of selection using CodeML . Ecol Evol 9 , 3891 – 3898 ( 2019 ). OpenUrl CrossRef PubMed 124. ↵ Minh , B. Q. et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era . Mol Biol Evol 37 , 1530 – 1534 ( 2020 ). OpenUrl CrossRef PubMed 125. ↵ Guindon , S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0 . Syst Biol 59 , 307 – 321 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 126. ↵ Lechner , M. et al. Proteinortho: Detection of (Co-)orthologs in large-scale analysis . BMC Bioinformatics 12 , 124 ( 2011 ). View the discussion thread. Back to top Previous Next Posted December 25, 2024. 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